Transient receptor potential (TRP) channels act as key regulators of many sensory systems including those for thermo-, photo-, and osmosensation [1-3]. A diversity of biological disorders has been linked to TRP channel malfunction ranging from neuropathic pain [4], cardiovascular diseases [5], to other diseases involving sensory physiology [6,7].

The TRP family of cation channels can be separated into seven subtypes according to structural similarity. Members of this family are nonselective cation channels that allow entry of extracellular calcium into cells resulting in a depolarization of membrane potential [8]. The mammalian TRPC (canonical) subfamily is proposed to function as store and second-messenger operated cation channels [9]. Disruption of TRPC may cause aberrant modulation of intracellular calcium and changes in membrane potential leading to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy. Currently there are few pharmacological modulators of the TRPC family and no compounds known to target specific TRPC isoforms [10].

The objective of the current screen is to identify compounds that block activation of TRPC4 by second-messenger signaling [11] within HEK293 cells that stably express this channel and mu-opioid receptor. Compounds selected as TRPC4 inhibitors will later be counter-screened for specificity.

Principle of the assay

To screen for compounds that inhibit the TRPC4 cation channel, a HEK293 cell line which stably expresses both the TRPC4beta and the mu-Opioid receptor is employed. Stimulation of the mu-Opioid receptor by DAMGO induces activation of TRPC4 through the G alpha signaling pathway. Channel activity is monitored by calcium flux with a commercial Fluo4 kit [11]. Compounds that show decreased Fluo4 fluorescence in the presence of the mu-Opioid receptor activator (DAMGO, in ECmax concentration) are considered inhibitor hits. mu-Opioid receptor inhibitors will be excluded through later counter-screening against HEK293 stable cells expressing the mu-Opioid receptor alone.

Assay overview: The objective of this assay is to identify compounds that inhibit the TRPC4 cation channel. A HEK293 cell line which stably expresses both the TRPC4beta and mu-Opioid receptor was employed, and channel activity was monitored by use of Fluo4, a cell-permeable calcium-sensitive dye. Compounds that inhibit TRPC4 result in a decrease in calcium flux in the presence of a mu-Opioid receptor agonist, DAMGO. This reduction in Ca2+ will result in a decreased fluorescence of Fluo4, as compared to control wells.The HEK293 stable cell line was seeded into 384-well plates. After overnight incubation, the cells were loaded with a calcium-sensitive dye, Fluo4, followed by an assay buffer wash. Cell plates were then loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader, where compounds were added and incubated for 110 seconds, before application of a submaximal concentration of DAMGO. Plates were again incubated for 110 seconds before application of DAMGO at a maximal activating concentration. Real-time fluorescence was then measured for 100 seconds. Library compound effect was evaluated by computing the integrated ratio of each well used for calculation and assignment of a B score for that well (B score Inhibitor Ratio, see Result Definitions, 1). Compounds that affected the fluorescence ratio within two seconds after application were flagged as fluorescent and removed from further analysis (Initial ratio, see Result Definitions, 2). Remaining compounds that scored less than the mean minus three times the standard deviation of the B score Inhibitor Ratio of all library members while also having a positive integrated ratio, were considered to be active as inhibitors of the TRPC4 cation channel.Protocol for the TRPC4 project:1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, 500 ug/mL G418 and 40 ug/mL hygromycin2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS.3. Incubate overnight at 37C and 5% CO24. Remove medium and add 20 ul/well of 1x Fluo4 solution to cells 5. Incubate 45 minutes at room temperature (RT) in the dark6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of DAMGO7. Remove Fluo4 dye solution and add 40 ul/well of assay buffer to cells8. Remove 40 ul solution and add 20 ul/well of assay buffer to cells9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader10. Measure fluorescence for 5 seconds at 1Hz to establish baseline11. Add 4 ul of 7.5x compound stock into the cell plates.12. Incubate plates for 110 seconds13. Add submaximal concentration of DAMGO and incubate for 110 seconds14. Add maximally activating concentration of DAMGO (DAMGO ECmax) and read for another 110 seconds.15. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout16. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [12]17. Calculate B scores [13] for test compounds using integrated ratios calculated in Step 1518. Outcome assignment: If the B score of the test compound is less than the mean minus 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds (B score Inhibitor Ratio 19. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Integer ((Log10(abs(B score Inhibitor Ratio))-0.7)*250); they are normalized to the smallest and largest LOG10(B score Inhibitor Ratio), B score Inhibitor Ratio, as in the result definition. The inactive test compounds are assigned a score of 0.List of reagents1. TRPC4 and mu-Opioid Receptor-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)2. PBS: pH7.4 (Invitrogen Cat#10010023)3. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma D5796-500ML)4. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)5. L-Glutamine (Invitrogen, Cat#25030081)6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)7. CellStripper (Mediatech 25-056-Cl)8. G418: (Invitrogen, Cat#11811-031)9. Hygromycin#(Mediatech, Cat#30-240-CR)10. HEPES (Sigma, Cat#H4034)11. 10XHBSS (#Invitrogen Cat#14065056)12. Pluronic F-127 (20% solution in DMSO) (Invitrogen Cat#P3000MP)13. [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) (Sigma Cat#E7384-10mg10MG) 14. Fluo-4 Calcium Assay Kit (Invitrogen, Cat # F14202)15. Triple-layer flask (VWR, Cat #62407-082)16. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)

Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust, in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary, based upon the actual sample provided by the MLSMR.